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LETlTERS TO THE EDITOR 2629

Fecal

Lactoferrin

as a

Marker of Fecal

Leukocytes

Weread with interest the report of Guerrant et al. (1). It was stated that the fecal lactoferrin assay may beauseful diagnos-tic tool in the evaluation of patients with acute diarrhea

syndrome (ADS), offering some advantages over microscopic

examination for fecal leukocytes. Wewould like to comment on this and report data from a study carried out in Chile in which fecal lactoferrin was measured as described elsewhere

(1) by using anti-human lactoferrin antibody (L-232; Sigma). Titers above 1:60wereconsidered positive.

In agreement with previous reports (1,2) showing that lactoferrin is present in human milk (1 g/liter), colostrum

(7g/liter), and cow milk (0.1 g/liter), we found lactoferrin-positive reactions at dilutions of up to 1:105 in milk samples from humans and cows and pasteurized liquid and infant formula(NANandNIDO;Nestle).Theantibodywasshownto cross-react with bovine lactoferrin,but onlywith human milk did itconsistentlyshowa more coarsepatternofagglutination.

The fecal lactoferrin assaywas evaluated with 63 pediatric patientswith ADS and 25 healthyinfants. Stoolsampleswere

collected for fecal lactoferrin,fecalleukocytes, and microbio-logical analysis (bacterial andparasitic entericpathogensand

rotavirus). The type of milk received 48 h prior to sample collectionwasalso recorded.

Among the 25 control infants, all were negative for fecal

leukocytes, 8 breast-fed infants were positive for fecal lacto-ferrin(5of themattitersover 1:540), and 17infants fed other kinds of milkwerenegative forfecal lactoferrin. The presence of lactoferrin in the stools appeared to correlate

strictly

with

breast-feeding. Since 10 of 63 patients with ADS had been

breast-fed, they were excluded from further analysis. Of 53

patientswith ADS, 28

patients

were

positive

for fecal

lacto-ferrin, 15 of themwere also

positive

forfecal

leukocytes,

and 22 produced samples that hadidentifiable enteric

pathogens

(enteropathogenic Escherichia coli

[EPEC], 11; rotavirus, 5;

Cryptosporidium

spp.,

3;

Campylobacter jejuni, 1;

Entamoeba

histolytica,

1;and

Shigella flexneri,

1).

Twenty-five patients

with

ADS were

negative

for fecal

lactoferrin,

23 of them were

negative for fecal

leukocytes

and

only

6 had an identifiable pathogen

(EPEC, 2;

rotavirus, 3;

and

Shigella

sonnei,

1).

These results suggest that fecallactoferrincould be a more

sensitive test than fecal

leukocytes

for the evaluation of

patientswithADS,butoneshould

keep

inmind the

possibility

offalse

positives

withbreast-fed

infants,

evenwhen

high

titers offecal lactoferrinare detected.

REFERENCES

1. Guerrant,R. L.,V.Araujo, E. Soares,K.Kotloff,A. A. M.Lima, W. H. Cooper,and A. G. Lee. 1992. Measurementoffecal lacto-ferrinasamarker of fecalleukocytes.J.Clin.Microbiol.30:1238-1242. 2. Sanchez,L., M.Calvo, andJ. H.Brock. 1992.

Biological

role of

lactoferrin. Arch. Dis. Child.67:657-661.

TeresaQuiroga PatriciaGarcia ManuelaGoycoolea

Marcela Potin LuisRodriguez

Pablo Vial

UDAClinicalLaboratoriesand

Department

of

Pediatrics SchoolofMedicine

Pontificia Universidad Cat6lica de Chile

Santiago, Chile

Author's

Reply

We

greatly appreciate

the

thoughtful

letter and additional information

by Quiroga

et al.

regarding

our report

(4).

In

further support of their

findings suggesting

that fecal

lacto-ferrin could bea more sensitivetestthan fecal

leukocytes

for

the evaluation of

patients

with acute diarrhea are several

recent additional reports from at least six different medical

centers

(1, 2,

6-9).

Specifically, Scerpella

etal.found that 94% oftravelerswith invasive

pathogens

had fecal

lactoferrin,

while

only

69% hadfecal

leukocytes (7).

Inaseparate report,

Yong

etal. noted that the fecal lactoferrin assaywas moresensitive

(75%)

than

methylene

blue

microscopy

(40%)

for the detec-tion of

leukocytes

in Clostridium

difficile

toxin-positive

fecal

specimens

(9).

Milleret al. noted

that,

in contrast to

healthy

volunteersand

patients

with classicalcholera

diarrhea, patients

with

shigellosis

or

enteropathogenic

E.

coli,

like those with

high-titer

C.

difficile

toxin,

typically

have a moderate if not

highly

inflammatory

process demonstrable

by

elevated fecal

lactoferrintiters

(6). Finally,

Croft et

al.,

Thorntonet

al.,

and

Andersonet al. all noted thatlactoferrinis 25to 114%better than

methylene

blue for

detecting

invasive

pathogens

(1,

2,

8).

Furthermore,

Thornton et al. noted that 33% of 21 stool

specimens

positive

for

lactoferrin

but

negative by

culture had

Shigella species

detected

by

PCR

(8).

While fecal lactoferrin is

highly

sensitive for an inflamma-tory process,

however,

the presence of fecal lactoferrin is

by

no means

specific.

Not

only

are many processes

capable

of

eliciting

an

inflammatory

enteritis

(including idiopathic

inflam-matorybowel

disease),

but

Quiroga

et al. raise the

potential

problem

of cross-reactions of certain anti-lactoferrin antibod-ies with human milk lactoferrin in the stools of breast-fed infants.

Fortunately,

while the lactoferrin found in

products

from other

species,

such as cow's

milk,

may show some

cross-reactionwithcertain antibodies suchasthe L-232

Sigma

antibody

used

by Quiroga

et

al.,

this is distinct from the

reaction seen with human

lactoferrin.

Although

we

certainly

share

Quiroga's

concern about this

possible

cause of

"false-positive"

fecal lactoferrin

determinations,

most diarrheal ill-nesses occurinchildrenafter

they

areweaned

(5),

andour own

experiences

with the anti-human lactoferrin

antibody

L-3262 from

Sigma Company

and with the Leuko-testas

prepared by

TechLab are different from theirs. We did not see

agglutina-tion whenwetested bovine milk. None of five human breast milk

samples

we tested gave lactoferrin latex

agglutination

titersof

>1:8;

threeshowed

agglutination

with the Leuko-test

beads

only

whenundilutedandwere

negative

ata1:2dilution

inbuffer.

However,

stillfurther dilutions showed that these five

milk

specimens

were

positive

at 1:100 and

1:1,000 dilutions,

suggesting

a prozone effect consistent with

(albeit

at lower

titers

than)

observations

by

Quiroga

and

colleagues.

Deris-bourg

et al. have

reported

the

significant

difference between the

glycans

of human

leukocyte

lactoferrin

and thoseof human

milk

lactoferrin,

with the human

leukocyte

lactoferrin

lacking

twofucoseresidues foundonhumanmilklactoferrin

(although

the authors

reported

no

antigenic differences) (3).

In the course of

checking

the Leuko-test kit with frozen human breast milk

samples

in our

laboratory,

we have also

found an

unexpected

coarse

agglutination

with the control normal

IgG-coated

beads when tested with human milk spec-imens. Whilethiswasnotseenwith thetest

antibody

(which

is

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2630 LETTERS TO THE EDITOR

clearly positivewith lactoferrincontrols),coarseagglutination with the control antibody as provided by TechLab might providea clue that breast milkmaybepresentin thespecimen. In conclusion, our experience and experiences in several

other laboratorieswouldcertainlyconcurwith that ofQuiroga andcolleagues; fecallactoferrin appearstobea moresensitive test than fecal leukocytes in the evaluation ofpatients with

acute diarrhea. However, as with any laboratory test, this

shouldbeinterpretedin the context of the clinicalpresentation (which, with fecal lactoferrin, may include the possibility of

false positives with breast-fed infantswhen tested with certain

antibodies).

REFERENCES

1. Anderson, D. E., J.Kramp, J.Claridge,M.B.Baker,E. M.Latimer,

and D. M. Lyerly. 1994. Detection and stability oflactoferrin in fecal leukocytes by latex agglutination compared to microscopic

evaluation, abstr.C-398,p. 560.Abstr.94thAnnu. Meet.Am.Soc. Microbiol. 1994.American Society forMicrobiology,Washington,

D.C.

2. Croft,S.,P.Newcomb-Gayman,and K.Carroll.1994.Comparison

oftheLeuko-test latexagglutinationassaywiththemethyleneblue stain for the detection of fecal leukocytes, abstr. C-396, p. 560. Abstr. 94th Annu. Meet. Am. Soc. Microbiol. 1994. American Society forMicrobiology,Washington,D.C.

3. Derisbourg, P., J.-M.Wieruszeski,J.Montreuil,andG.Spilk 1990. Primarystructureofglycans isolated from humanleucocyte lacto-transferrin.Biochem.J.269:821-825.

4. Guerrant, R.L., V.Araujo,E. Soares,K. Kotloff,A.A. M.Lima,

W.H.Cooper,andA. G. Lee. 1992. Measurementoffecal lacto-ferrin as a marker of fecalleukocytes.J.Clin.Microbiol. 30:1238-1242.

5. Guerrant,R. L.,L. V.Kirchhof, D. S.Shields, M. K. Nations,J. Leslie,M.A. de Souza,etal. 1983. Prospective studyof diarrheal illnesses in northeastern Brazil: patterns of disease, nutritional impact,etiologies, and risk factors. J. Infect. Dis. 148:986-997. 6. Miller,J. R.,L.Barrett, K. Kotlof, and R. L. Guerrant.

Antilacto-ferrinantibodylatex beadagglutination assayas arapiddiagnostic

testforinflammatory enteritis. Arch. Intern. Med., in press. 7. Scerpella,E.G., P. C. Okhuysen, J. J. Mathewson, R. L.Guerrant,

E. Latimer, D. Lyerly, et al. 1994. Evaluation of a new latex agglutination test for fecal lactoferrin in travelers' diarrhea. J. Travel Med. 1:4-7.

8. Thornton, S.,T.O'Brien, J. Callahan, T. Sharp, R. Batchelor, P. Rozmajzl,M.Wallace,andJ. Burans. 1994. Evaluation ofarapid latex testfordetectionof lactoferrin instool, abstr. C-397,p.560. Abstr. 94th Annu. Meet. Am. Soc. Microbiol. 1994. American Society forMicrobiology, Washington, D.C.

9. Yong,W.H.,A. R.Mattia,and M.J. Ferraro. 1994. Comparisonof fecal lactoferrin latex agglutination assay and methylene blue microscopy for detection of fecal leukocytes in Clostridiumdi ffcile-associateddisease. J. Clin.Microbiol.32:1360-1361.

Richard L.Guerrant,M.D. ClovisMartins,M.D. Terezinha Silva,M.D.

Divisionof Geographicand International Medicine Charlottesville, Virginia22908

Use of Selective Media

for

Isolating Corynebacterium urealyticum

from

Urine

Specimens

Corynebacteriumurealyticum, formerlyknownas Corynebac-teriumgroupD2,is a newspeciesin thegenusCorynebacterium (3)which has been involvedmainlyinurinarytractinfections but also inendocarditis, pneumonia,peritonitis, osteomyelitis,

and soft-tissue infections (1, 5, 6).The clinical significance of

mostorganismsisolatedfromnormallysterilesitessuchasthe

kidney,the bladder wall, the ureter, and blood is relatively easy

to determine. However,that of organismsisolated from non-sterile areas, suchasurine or sputum, is moredifficulttoassess. Ryan and Murray (4) have recently examined thevalue of selective media for isolation of C. urealyticum from urine samplesas well as determination of the clinical relevance of such isolates.They studied 194 urine samples which had pHs of

.7.0,

findingtwoisolates ofC. urealyticum (prevalence, 1% in

urine samples with such a pH) which were not related to the

urinarytractinfectin symptoms. This finding partially confirms those of a previously published paper which included more than9,000 unselected urine samples showing a prevalence of 1.17% with selective media but only 0.038% with nonselective media (7). Unsurprisingly, most or all of the 15 organisms isolated from 13 patients by using selective media were not

involvedin the clinical symptoms, but those isolated from three patients by using nonselective media were (7). De Briel et al.

(2), by studying more than 5,000 unselected urine

samples,

isolated C.

urealyticum

in 2.5% (2105 CFU/ml) of the urine samples using selective and nonselective culture media (2). Our rates of C. urealyticum isolation from unselected urine samples are 1.9 and 0.23% with selective and nonselective

media,respectively. Again, up to 60% of isolates from

nonse-lectivemedium were clinically significant (5); the rate for those

isolates from selective medium was very low (unpublished data). Selectivemediumforisolating C. urealyticumfromurine sampleshasgreatepidemiological value,but itisnotuseful for

managementof thepatients,as moststrains isolatedonly from selective medium have noclinicalrelevance.

The decision to lookfor C. urealyticum in urine specimens and therefore to extend the incubation of urine cultures is a matter open for discussion. Nevertheless, we do not recom-mend the use of selective media for routine purposes. We

recommend cystine lactose electrolyte-deficient and blood

agars instead. Severalcircumstances, such as kind of hospital,

local prevalence, data from urine sediment, and above all, clinical information communicated to the microbiologist, as

alsostatedby Ryan and Murray(4), should be considered key factorsbefore onedecides to search for this pathogen.

REFERENCES

1. Coyle, M. B., and B. A. Lipsky. 1990. Coryneform bacteria in infectious diseases:clinical and laboratory aspects. Clin. Microbiol. Rev.3:227-246.

2. De Briel,D., J. C. Langs, G.Rougeron,P.Chabot, and A. Le Faou. 1991. Multiresistant corynebacteria in bacteriuria: a comparative studyof therole of

Corynebacterium

group D2 andCorynebacterium jeikeium. J. Hosp. Infect. 17:35-43.

3. Pitcher, D., A. Soto, F. Soriano, and P. Valero Guillen. 1992. Classification ofcoryneform bacteria associated with human uri-nary tractinfection(group D2) asCorynebacteriumurealyticum sp. nov. Int. J.Syst.Bacteriol.42:178-181.

4. Ryan,M., and P. R. Murray. 1994.Prevalence ofCorynebacterium urealyticum in urine specimens collected at a university-affiliated medical center. J. Clin.Microbiol. 32:1395-1396.

J. CLIN. MICROBIOL.

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Vol.30,No. 5 JOURNALOFCLINICALMICROBIOLOGY, May1992,p. 1238-1242

0095-1137/92/051238-05$02.00/0

Measurement

of Fecal

Lactoferrin

as a

Marker of Fecal

Leukocytes

RICHARD L. GUERRANT,l* VALTERARAUJO,1 ELIZABETHSOARES,"2 KARENKOTLOFF,3 ALDO A. M. LIMA,2 WILLIAM H.COOPER,' ANDAMELIA GAIL LEE'

DivisionofGeographic Medicine, Department of Medicine, University of Virginia SchoolofMedicine, Charlottesville, Virginia 229081; Center for VaccineDevelopment, University of Maryland, Baltimore, Maryland212283;

and Clinical Research Unit, FederalUniversity of Ceard, Fortaleza, Ceara, Brazil2

Recieved 12 November 1991/Accepted 31January1992

While diarrheal illnesses are extremely common in communities and hospitals throughout theworld, an

etiologic diagnosismaybeexpensiveand cost-ineffective.Althoughthepresenceof fecalleukocytesarehelpful

inthediagnosisandspecific therapy ofinflammatory diarrheas, this requirespromptmicroscopicexamination offecal specimens (preferably obtained in a cuprather than a swab ordiaper) by a trained observer. We

developed a simple, sensitive test for the detection of leukocytes in fecal specimens using antilactoferrin

antibody. Whereas radial immunodiffusion detected 0.02,ugoflactoferrin (LF) per ,ulor .2,000 leukocytes per,ul, latex agglutination (LA) readily detected .0.001 ,igof LFper,ulor .200leukocytesper,ul addedto

stoolspecimens. Despitethe destructionorloss ofmorphologic leukocytesonstorage for1to7daysat4°Cor

placementofspecimenson swabs, measurable LF remained stable. Initial studies of stoolspecimensfrom six

patients with Salmonella orClostridium difficile enteritis were positive and those from three controls were negative forLF by LA.Of 17 children in Brazil withinflammatory diarrhea (.1 leukocyte perhigh-power field), 16 (94%) had LF titers of >1:50 byLA,whereasonly3 of12fecal specimens with <1leukocyte per high-power field on methyleneblue examination and noneof 7 normal control specimens had anLFtiterof

>1:50byLA. Of16 fecalspecimensfrompatientswithC.

difficile

diarrhea (cytotoxin titers, .1:1,000),95%

(n = 15)haddetectable LFbyLA(intiters of 1:100to1:800). Finally,of 48 fecalspecimensfromhealthyadult

U.S. volunteers before and after experimental shigellosis and of 29 fecal specimens from children with

documentedshigellosisand hospitalizedcontrols innortheastern Brazil,fecal LF titers rangedfrom 1:200to

>1:5,000in96%(25of26) samplesfrompatientswithshigellosis (and reported positiveforfecalleukocytes), while51 controlsconsistently had fecalLFtitersof<1:200.We concludethatfecal LF isauseful markerfor fecal leukocytes, evenwhen theyare morphologicallyloston swabspecimens orwhenthey aredestroyedon

transportorstorageorby cytotoxicfecal specimens.

Diarrheal illnesses areextremelycommon

throughout

the

world,causing2to 16or moreillnesses per person per year in developed and developing countries (7,

15)

and often

posing diagnostic and therapeutic questions forphysicians.

The causes of diarrhea include awide variety of etiologic

agents, manyof which have beenrecognized onlyinthe past

two decades (7). However, these agents do notneed tobe

exhaustivelysoughtin everyinstance of thiscommon

prob-lem (6, 23), and the cost of indiscriminate use of etiologic

studies for diagnosis isprohibitive. The costfor each

posi-tiveroutine stool culture result has exceeded

$900

to

$1,000

(8, 11).

An important diagnosticclue inconsideringwhether diar-rheaisanoninflammatoryor aninflammatoryprocessis the examination for fecal leukocytes (4, 9, 12). If fecal leuko-cytes are present, they suggest an inflammatory process caused by Salmonella species, Shigella species,

Campylo-bacterjejuni,

orClostndium

difficile.

Although the majority of cases are noninflammatory (rotaviruses, Norwalk-like

viruses, enterotoxigenic

Eschenichia

coli) and often respond

tosimpleoral rehydration therapy, it is important to distin-guish theinvasive,inflammatorydiarrheal illnesses that may be more severe and that should be the focus of more extensivediagnostic studies and cultures and/or antimicro-bial therapy (8). However, the methylene blue examination for fecal leukocytes requires that the physician or a skilled

microscopist promptly examine under a microscope mucus

*Correspondingauthor.

from a fecal specimen in a cup. Thespecimen is stained so

thatleukocytes are clearly distinguishable in the fecal debris. This requires the immediateavailability of a skilled person with a microscope to stain and examine fresh fecal speci-mensin the clinic or emergency areas where the patient is seen. It may also bedifficult to obtain fecal specimens in a

cup(these specimens aresuperior to swabor diaper speci-mens for examination of fecal leukocytes; swab or diaper specimens are only 44% sensitive, whereas cup specimens are95% sensitive for fecalleukocyte detection by methylene blue examination of specimens from patientswith culture-documented shigellosis [12]). Therefore, a method that

re-mains sensitive when swabs are used and that remains sensitive when specimens are transported or stored (over-night) would behelpful and might allow a wider application ofaselective diagnosticand treatment algorithm.

Wedevelopedasimple in vitro test for a leukocyte marker that is highly sensitive to the numbers of fecal leukocytes

typicallyfound ininflammatorydiarrheal specimens and that

can be quicklyand easily done either in the clinic or later (after transportation or storage) in the laboratory. Only a minimum amount of training is required to learn how to perform the test.

We initially explored the leukocyte marker leukocyte

esterase (an enzyme used in detecting leukocytes in the urine) (16) and found that, in contrast to its usefulness in testing leukocytes in urine, normal fecal specimens gave a

positive result,so weabandoned further studies with leuko-cyte esterase. Instead we found that lactoferrin, an

iron-binding glycoproteinfound concentrated in secondary

(4)

MEASUREMENT OF FECAL LACTOFERRIN 1239

ules in leukocytes (10, 19), was not readily detected in normal stoolspecimens unless neutrophils were added. The neutrophils were then readily detected in fecal specimens when lactoferrin was used as a marker. While we initially demonstrated the feasibility of detecting leukocytes in fecal specimens using a radial immunodiffusion assay for lacto-ferrin, the greater sensitivity and speed of latex agglutination led to a focus on that method, as noted below.

MATERIALSANDMETHODS

Preparation of latex beads. Latex beads(Bacto-Latex 0.81 beads;Difco Laboratories, Detroit, Mich.) were coated with rabbit anti-human lactoferrin (product L-3262; Sigma Chem-ical Company, St. Louis, Mo.) as follows: 2.5 ml of beads wascentrifuged at 1,800 xgfor 30min, washed with 5 ml of glycine buffer (7.3 g of glycine and 10 g of NaCl in 1 liter of distilled water adjusted to pH 8.2 to 8.3), and then

resus-pendedin5 ml ofglycine buffer to provide an approximately

1% suspensionof beads. To this latex bead suspension was added 0.35 ml of undiluted rabbitantilactoferrin antibody, to

providea7% antibody dilution in the bead suspension. The mixture was incubated at 38°C for 1 h, after which the

antibody-coatedbeads were spun and resuspended in5ml of buffertowhich 0.005 g of azide(0.1%) and 0.05 g of bovine serum albumin (1%) were added. The coated bead suspen-sion was then stored at 4°C until use. Twenty microliters of thisantibody-coated latex bead suspension was mixed on a microscope slide with 20 ,ul of sample, andagglutination was graded after 2 min with an unaided eye as follows: 0, no

agglutination; +,barely detectable, trace agglutination with

amilky background; 1+, definite, fine agglutination with a milky background; 2+, definite, fine agglutination with a clearing background; 3+, larger agglutination with a clear

background.

For a negative control, latex beads were prepared as described above, but rabbit anti-human lactoferrin was not added. The assay has been licensed and is being developed

as acommercialproductby Techlab Inc., Blacksburg, Va. (a patentispending).

Isolation of neutrophils. Neutrophilswere obtained from normal heparinized (10 ml) venous blood by a one-step Ficoll-Hypaque separation procedure (Neutrophil Isolation

Medium; Los Alamos Diagnostics, LosAlamos, N.M.)(3). The polymorphonuclear leukocytes (PMNs) were washed three times with Hanks balanced salt solution (HBSS).

Residualerythrocyteswere lysed byhypotonic lysiswith 3 mlof iced 0.22%sodiumchloride solution for 45s andthen with 0.88 ml of 3% sodium chloride solution, this was

followedby theadditionof 5 ml ofHBSSand

centrifugation.

A1:2suspensionof PMNs in diluted stoolwas

prepared

by using normal stool of mixed with 1 ml of HBSS to make a cloudy suspensionand

adding

0.5 ml ofpreviouslycounted PMNsto0.5 ml of the stool

suspension.

A1:2suspensionof PMNs in HBSSwas

prepared

as a control for

comparison

with fecal

suspensions.

To standardize our

antibody-coated

latex bead prepara-tion,wedetermined dose-responsecurves

using

asolution of

lactoferrin (L-0520;

Sigma)

diluted in HBSS.

Testing of PMNs in fecal specimens after

refrigeration

or placement on a swab. To test for PMNs in specimensthat

were

refrigerated

or

placed

on

swabs, aliquots

of

previously

countedPMN-stool

suspensions

were

refrigerated

or

placed

on swabs for the indicatedtimes. For swab

specimens,

130 ,ul (we found that this volume saturates the

swab)

of a

previously counted PMN-stool

suspension

was usedto

sat-3+- 1

2+o

Lactoferrin 1+ Latex

Agglutination

.08 .15 .31 .62 1.25 2.5 5 10

Lactoforrin (ng/pi)

FIG. 1. Sensitivity of the latex agglutination assay for lactofer-rin. Data represent the mean (line) and individual results from four different experiments (0) by using two different preparations of lactoferrin and three different preparations of antibody-coated beads.

urate a rayon-tipped swab (Culturette II collection and transportsystem;MarionScientific,Kansas City, Mo.). The swabwasthenplaced in 390 ,u of HBSS and shaken, and the excess fluid was squeezed out to make a calculated 1:4 dilution that we could examine for leukocytes directly and by thelactoferrin assay.

PMNswerequantified morphologically at time zero and at subsequent intervals by using a ruled Neubauer-type hema-cytometer chamber. To aid in visualizing the nuclei,

meth-ylenebluestainwasincorporatedintothecounting solutions (20 ,u of solution, 20

ptl

ofmethylene blue, 160 ,ul of HBSS).

RESULTS

Sensitivity

oflactoferrin latexagglutination assay for

lacto-ferrin. Asshownin Fig. 1, the lactoferrin latex agglutination assaydonewith three different antibody-coated bead prep-arations wassensitive to less than 1 ng of purified lactoferrin per,ul, withreadily apparentagglutination of the latex beads. The lowest concentration of

lactoferrin

that consistently

gave 1+ or greater agglutination was 0.31 ng/,u, a concen-tration that, from previously published data (10), would be

expected to be present in 60 PMNs per ,u or60 PMNs per

mm3,

a number much lower than that innormal peripheral

blood and substantially lower than that expected in an

inflammatory fecal specimen.

Sensitivityoflactoferrin latexagglutinationassayfor PMNs in HBSS or stool suspensions. By using human PMNs that were separatedbyFicoll-Hypaque andsuspended in HBSS

ornormal stoolsuspensions,thesensitivityof thelactoferrin

latex agglutination assay that gavedetectable trace

aggluti-nationwas60to 140 PMNs per

RI,

and instool

specimens,

120to280 PMNs per

RI

gavedefinite 1+to2+

agglutination

(Fig. 2), with slightly greater

sensitivity

seen when the

PMNs were

suspended

in stool

specimens

than when

they

were

suspended

in HBSS. The stool

specimens,

like the

detergent TritonX-100

(0.1%),

therefore

appeared

torelease lactoferrin. Therewas no further increase in the

sensitivity

forPMNs in fecal

suspensions

when 0.1or 1% Triton X-100

wasused. This number of PMNswasin the range

expected

from the assay

sensitivity

for lactoferrin

and,

again,

was

substantially

below that which would be

expected

from

microscopic

examination of

inflammatory

fecal

specimens.

To compare the

feasibility

of

detecting

leukocytes

by

morphologic

counts or

by

latex

agglutination

forlactoferrin

(5)

1240 GUERRANT ET AL.

/- , ,,---...

Lactoferrin 1+,

Latex

Agglutination

|,.x_x

0-

*

~~~~~~~~~HBSS

0.1%Triton

0 100 200 300 400 500 600 700 800

Calculated PMN/4I

FIG. 2. Sensitivity of the lactoferrinlatexagglutinationassayfor PMNs in HBSS or stool suspensions. Each line represents a

separate experiment. Stool suspensions(likeTriton X-100) appear to release lactoferrin and, thus, increase the sensitivity of the lactoferrin latexagglutination assay.Thesensitivityof the lactofer-rinlatexagglutinationassaythatgavedefinitefineagglutination(1+) was alactoferrincontentof 120to240PMNsper,ul.

after storage in a refrigerator or on swabs, the number of

PMNs inthesuspensionswerecounted and then thePMNs

wereplacedin1% Triton X-100orinacloudy

suspension

of

normal stool orboth.Then, we reexamined thesuspension

forPMN counts by

microscopy

and lactoferrin titer

by

the

latexagglutination assay

immediately

and after storage in a refrigerator or on swabs for 1 to 6 days (Fig. 3 and 4). In contrast tothe labilityofPMN numbersand morphologies seen after refrigeration, lactoferrin titers were remarkably

stableevenwhen thestoolorTriton X-100

suspensions

were refrigerated for severaldays.

There was a striking loss of PMN numbers after PMNs

were placed on swab specimens

(Fig. 4).

In contrast, as quantifiedbydeterminingthe lactoferrin titer ofleukocytes

in fecal suspensions placed on swab specimens remained

stable. Lactoferrin titerswere

relatively

stable afterPMNs were placed in a fecal suspension and then

placed

on rayon-tippedswabs(incomparisonwith the lactoferrintiters

thatweredetermined beforeplacementof the

suspension

on swabs). Incontrast, the number of

morphologically

evident leukocyteswasextremely

variable,

withameanloss of 3to 4 log units afterplacementon aswab for 1to 6days.

Clinical studies. Initial

pilot

studies of stool specimens

fromsixpatientswithSalmonella

(n

=

2)

orC.

difficile

(n

=

1:1600

1:800

Lactoferrin

Latex 1:400

Agglutination (LFLA titers)

(-) 1:200

1:100

T

It

--I., . T+S S

it It

it

T+S

X b

s ".

., s~~~~~~0

-1 0 1 2 3 4 5

Days

*107

,o6

-10

3

,<10

7

FIG. 3. Stability of lactoferrin latex agglutinationassay(LFLA)

titers after refrigeration of specimens for 1 to 6 days. Each line representsaseparateexperiment. Incontrasttothelabilityof PMN

numbers andmorphologiesseenin paired experiments, lactoferrin

titers wereremarkably stable, evenwhen solutionswere

refriger-atedforseveraldays. T, 1% Triton X-100; S, stoolspecimen.

Log

LFLA Titer

(-)

5 Mean

log

.4 PMN/4I (±range)

33

Stool: PMN Suspension

FIG. 4. Stability of lactoferrin latex agglutinationassay(LFLA)

titersdespite theinstabilityof PMNcountswithswabspecimens in

1 to 6 days. "Alone" indicates PMNcounts andlactoferrin titers determinedin thefecal-PMNsuspension just before their placement ontheswabs. Lactoferrinwasconsistently detected in swab spec-imenswithminimalornoloss, incontrast tothestriking variation with a mean 3- to4-log-unitlossofmorphologicPMNcountsafter placementonswabspecimens. Resultsarethemeansandrangeof three and four different experiments for PMN and lactoferrin

measurements,respectively.

4) enteritis were positive and three control stool specimens

were negative for lactoferrin by latex agglutination, thus

demonstrating the feasibility of using this test to examine patient fecal specimens. In preliminary studies to test whether the lactoferrin latexagglutinationmethod correlated withevidence of fecal leukocytesonmicroscopy of methy-lene blue-stained specimens in a field setting, fecal speci-mensin cupsfrom 29 children withacutediarrheapresenting

to aRehydration Center inFortaleza, Ceara, Brazil, where stool cultures are not routinely available, were promptly

examined. Of 17 children whosespecimenshadone ormore leukocytes per high-power field on microscopic examina-tion,wedeterminedbylatexagglutinationthat 16(94%)had fecal lactoferrin at a titer of greater thanorequalto 1:50. In only 3 of 12 children (25%) whose specimens had less than

oneleukocyte perhigh-powerfieldwaslactoferrin presentat

atiter of greater than orequal to 1:50. Inaddition, noneof the seven normal controlspecimensfromhealthyindividuals without diarrhea in Charlottesville, Va., was positive for

lactoferrinat atiter of greaterthanorequalto1:50. When we

systematically examinedbycellculture assay 16 fecal spec-imens frompatientswith nosocomialdiarrheaatthe

Univer-sityofVirginiaHospital that had C.

difficile

cytotoxintiters greater thanor equal to 1:1,000, wefoundbylatex aggluti-nation that 95% (15 specimens) had lactoferrin titers that

ranged from 1:100 to 1:800, even though PMNs were not

seen in 6 of 9 of these specimens that were promptly examined after methylene blue staining. Of 14 fecal

speci-mens with cytotoxin titers of 1:10 to 1:100, 9 (64%) had moderatelactoferrintiters(1:100to1:400), while only 1 of 16

specimens (6%)with nodetectablecytotoxin had a lactofer-rin titer of >1:50(1:100).

Finally, we tested fecal specimens from patients with

culture-documentedshigellosis from the University of Mary-land Center for Vaccine Development and Hospital das ClinicasinFortaleza,Ceara, Brazil (Fig. 5). In contrast to 0 of 34 fecal specimens from healthy control adults in the United States that hadatiter of >1:50 or 0 of 17 hospitalized children without diarrheain Fortaleza with fecal lactoferrin titers of>1:200, in 25 of 26 (96%) fecal specimens from adult

J. CLIN. MICROBIOL.

(6)

MEASUREMENT OF FECAL LACTOFERRIN 1241

1:6400

1:3200

1:1600'

1:800 1:400 Lactoterrin 1:200

Latex Agglutination1:100

1:50

1:25 1:12.5

1:6.25

<1:6.25

A

.A AA

A&

AAAAiAAaA

L*L*LA

*

Pro-inmsetn Post (n=34)

Healthy U.S. Vol

a

A

AAA

A

aaLAAA

AAAA

A

,.o.p.

.f..o- an*gu**o

(nt14) Contol Ca$"

(n.17) (n-12)

unteers HospitalizedChildren In Brazil

FIG. 5. Fecal lactoferrin titers in controlsandpatientswith both experimentaland naturally occurringacute shigellosis. (A) Lacto-ferrin latex agglutination assay titers in the stools of34 different

healthy adult volunteers before experimentalchallenge in vaccine studies and in 14 individuals who developed acute inflammatory diarrhea after experimentalShigellaflexneri infection. (B) Lacto-ferrin latexagglutinationassaytiters in thestools of 12 children with

acute, culture-documented shigellosis and 17 hospitalized control children withoutdiarrheainFortaleza, Ceara,Brazil.

andpediatric patients with documented shigellosis with fecal leukocytesonexaminationby methylene blue staining, fecal

lactoferrin titerswere 21:200 (rangingto 21:5,000).

DISCUSSION

Results of the studies described here demonstrate the practical feasibility of using latex agglutination for determi-nation of fecal lactoferrin as a simple, semiquantitative

marker for PMNs in fecal specimens. Lactoferrin latex agglutination provided a highly sensitive means to quantify PMNs in fecalspecimens. It detected lactoferrinat<1ng/,ul

and <200 PMNsper ,ul, anumber far less thattheexpected number of PMNs ininflammatory fecal specimens. Although fecal leukocytes have long been recognized as being of

diagnostic value, there is little informationon theexpected number of PMNs ininflammatory fecal specimens. Reported numbers ofleukocytes have ranged from multiple cells on

fiveormorehigh-power fields (18)ortwoor moreleukocytes per high-power field seen in 86 to 91% of patients with

Shigella, C. jejuni, and Salmonella infections (versus 0 to

2% for patients with viral, Giardia, or toxigenic bacterial

diarrhea) (1)to >50leukocytesperhigh-powerfieldreported in fecalspecimensfrom 39to85% ofpatientswithshigellosis (20, 21). Speelmanetal.(20) noted that themeannumber of

leukocytespercubic millimeter determinedby

hemacytom-etercounts was28,700 + 4,300for 33 patientswith

shigel-losis (correlated with a mean of 81.6 ± 6 leukocytes per

high-power field). Therefore, oneleukocyteper high-power field would equate to approximately 350 leukocytes per mm3.Therefore,our1:50 dilutionwouldrepresenta

thresh-oldofapproximately8 to 30leukocytesperhigh-powerfield

(basedon thesensitivityofourtestof 60to 200leukocytes per .ul). Lactoferrinis foundinspecific (secondary) granules

in PMNs and isnotfoundinlymphocytesormonocytes (5,

13, 14). Although lactoferrin is present in several bodily secretions,the amounts range from 4.7to 26

ng/pul

in saliva (22)to3.8to218 pig/mgofproteininvaginalmucus

(depend-ingonthe time from the patient's lastmenses

[2])

and are

substantiallylowerthan the amountsof lactoferrinexpected fromthe number ofleukocytes typicallyseeninpatientswith

recognized inflammatory enteritis. At our 1:50 screening

dilution,wemaydetectaslittleas 15 ng oflactoferrinper

,ul,

or about 3,000 PMNs per

Rl.

In our pilot field studies in

Brazil, this correlated with 21 PMNper high-power field.

Although Stoll et al. (21) noted that 99% of 304 stool

specimens from patients infected with Shigellaspecies,96% of262 stoolspecimens frompatients infected withC jejuni,

and 98% of 137 stool specimens from patients with

Ent-amoeba

histolytica

showed >1 leukocyte per high-power field, 20to29% ofpatients with rotaviral, enterotoxigenicE.

coli, and cholera (all presumably noninflammatory diarrhea)

had 11 to20leukocytesperhigh-powerfield, suggesting that the thresholdseparating patients with primary inflammatory diarrhea from thosewithnoninflammatory diarrhea maybe higher in areaswheremultiple bacterial and parasitic infec-tions are common. Harris et al. (9) noted >25 PMNs per

high-power

field in68% of patients with

Shigella

orinvasive

E. coli colitis.

Lactoferrin was stable in fecal specimens, even after transportation, storage, swab, ortoxindestroyed the

leuko-cyte

morphology. Preliminary experience

showed that in

patients

with

inflammatory

diarrheas causedby Salmonella

species

and C.

difficile (cytotoxin positive,

antibiotic-asso-ciated

diarrhea),

lactoferrin was

readily

detectable by this

method. Fieldstudiesshowed that for94%ofpatients(16of 17 patients) with fecal specimens with one tofive or more

fecalPMNs per

high-power

fieldon

methylene

blue

staining,

lactoferrin latex

agglutination

titerswere >1:50. According

tothe

sensitivity

ofourassay, thiscorresponds to3,000to

12,000 PMNs per

RI,

a concentration range that would be

expected

to correlate with one or more PMNs per

high-power field in a blood smear. Furthermore, we found a correlation ofC.

difficile

toxin titers with lactoferrin positiv-ity, despite an apparent lack of morphologically evident PMNs in many of the toxin-containing stools. Finally, in contrast to none of 51 controls (34 healthy U.S. adult volunteers and17childrenhospitalized with diagnosesother thandiarrheainFortaleza)who had fecal lactoferrin titers of

>1:200, 96%

(25

of

26)

of

specimens

from

patients

withacute

shigellosis (under

experimentalorfield

conditions)

and fecal

leukocytes reported on initial examination by methylene

blue

staining

had fecal lactoferrin titers of

21:200.

Theone

specimen reported

oninitial examinationtohave

leukocytes

but low lactoferrin concentrations was

unconfirmed,

be-cause examination of all the frozen

specimens

failed to

revealleukocytes,

despite

theirpresence in

large

numbers in the initial examination ofmost

specimens.

A

possible

expla-nation for this includes a

false-positive reading

of the wet mountfor fecal

leukocytes

(false-positive

wet mount

read-ings

are well documented when fresh fecal

specimens

are

also fixed for

subsequent

staining

and examination

by

oil-immersion

microscopy by

additional trained

microscopists

and for

methylene

blue

staining

and

lactoferrin

studies).

Alternatively,

frozen fecal

specimens

may not

always

pre-serve lactoferrinor an inhibitormay

rarely

bepresent. The

controlsshow

that,

whereas

healthy

adults who reside in the

United States

consistently

have fecal

lactoferrin

titers of

c1:50,

childrenwithout diarrheamay havefecal

lactoferrin

titers of as

high

as

1:200,

perhaps

reflecting

a subclinical

inflammatory

responseto a commonenteric

parasite

suchas

Ascaris

species,

Tichuris

species

or hookworm

(17)

or to subclinical

malabsorption

of

milk,

salivary,

or serum pro-tein. In this

setting,

the fecal lactoferrin test may

help

to

define mild subclinical

inflammatory

enteritis,

and the

threshold for

highly

significant inflammatory

enteritis may thus be

interpreted

more

cautiously, perhaps

witha

(7)

1242 GUERRANT ET AL.

line zone oftitersrangingfrom 1:50to 1:200suggestingmild inflammation or protein malabsorption. While breastfed in-fants are much less likely to develop serious diarrhea, we

testedstoolspecimens from four healthy breastfed infants in Charlottesville,Va., in whom lactoferrin titers ranged from <1:50 to 1:100. Additional studies with larger numbers of

culture-documented inflammatory and noninflammatory eti-ologies of diarrhea are needed. However, the lactoferrin latexagglutinationtestcorrelates with evidence of PMNs in fecalspecimensdetermined bymethylenebluestaining,and lactoferrinremainsintactevenwhen theleukocyte

morphol-ogyisdestroyedontransferorstorageorby cytotoxicfecal

specimens. Furthermore, it is ofconsiderable practical im-portance that the lactoferrin assay can detect PMNs in specimensobtainedon swabs that adsorb most

morpholog-ically detectable PMNs.

In conclusion, fecal lactoferrin canreadilybedetectedby

a simple latex agglutination assay and provides a useful

marker for inflammatory diarrhea, even when fecal

leuko-cytes are destroyed by transportation, storage, swab, or

toxins. The ultimate utility of this assay in clinical or field settingsand itspotential applicationtospecimensotherthan

stoolspecimens require prospectiveclinical studies in differ-entfield, hospital, clinical, andlaboratorysettings.

ACKNOWLEDGMENTS

This workwassupportedin partbyaUSAID grant fromDiaTech, PATH,Seattle,Wash.,and in partbyaUSAID/CNPQpostdoctoral

fellowshipfor V.Araujo.

We appreciate the technical assistanceof Leah Barrett and the helpof Yatta Jacob inpreparing the manuscript.

REFERENCES

1. Alvarado, T. 1983. Faecalleucocytesinpatientswithinfectious diarrhoea.Trans. R. Soc.Trop. Med.Hyg. 77:316-320. 2. Cohen, M. S., B. E. Britigan,M. French, and K. Bean. 1987.

Preliminary observations on lactoferrin secretion in human vaginalmucus:variationsduringthemenstrualcycle,evidence of hormonal regulation and implications for infection with Neisseria gonorrhoeae. Am. J. Obstet. Gynecol. 157:1122-1125.

3. Ferrante, A., and Y. H. Thong. 1980. Optimal condition for simultaneous purificationof mononuclear and polymorphonu-clear leukocytes from human blood by the Hypaque-Ficoll method. J.Immunol. Methods 360:109-117.

4. Grady,G. F., and G. T. Keusch. 1971.Pathogenesisofbacterial diarrheas. N. Engl.J. Med.285:831-841.

5. Green, I., C.H.Kirkpatrick, and D. C. Dale. 1971. Lactoferrin-specificlocalization in the nuclei of humanpolymorphonuclear neutrophilicleukocytes. Proc. Soc. Exp. Biol. Med. 137:1311-1317.

6. Guerrant,R. L.1988.Campylobacterenteritis, p. 1648-1651. In J. B.Wyngaarden (ed.),Cecil textbook of medicine. The W. B. SaundersCo.,Philadelphia.

7. Guerrant,R.L., J. M. Hughes, N.L.Lima, and J. K. Crane. 1990. Diarrhea indeveloped and developingcountries: magni-tude,special settings, and etiologies. Rev. Infect. Dis.12(Suppl. 1):S41-S50.

8. Guerrant, R. L., D. S. Shields,S. M. Thorson, J. B. Schorling, and D. H. M.Groschel. 1985.Evaluation anddiagnosisof acute infectious diarrhea. Am. J. Med. 78:91-98.

9. Harris, J. C., H. L. DuPont, and B. R. Hornick. 1972. Fecal leukocytes in diarrheal illness. Ann. Intern.Med. 76:697-703. 10. Hetherington, S. V., J. K. Spitznagel, and P. G. Quie. 1983. An

enzyme-linked, immunoassay(ELISA)for measurement of lac-toferrin.J. Immunol. Methods 65:183-190.

11. Koplan, J. P., H. V. Fineberg, M. J. B. Ferraro, and M. L. Rosenberg.1980. Value of stool cultures. Lancet ii:413-416. 12. Korzeniowski, 0. M., F. A. Barada, J. D. Rouse, and R. L.

Guerrant. 1979. Value ofexamination forfecalleukocytes in the earlydiagnosisofshigellosis.Am.J. Trop. Med. Hyg. 28:1031-1035.

13. Leffell, M. S., and J. K. Spitznagel. 1972. Association of lactoferrinwith lysozyme ingranules of human polymorphonu-clearleukocytes. Infect.Immun. 6:761-765.

14. Masson, P. L., J.F. Heremans, and E. Schonne. 1969. Lacto-ferrin, an iron-binding protein in neutrophilic leukocytes. J. Exp. Med. 130:643-658.

15. Mata, L. J., and R. L. Guerrant. 1988.Magnitude and impact of diarrhoeal disease, p. 435-445. In R. L. Guerrant (ed.), Bail-liere's clinical tropical medicine and communicable diseases. BailliereTindall, London.

16. Oneson, R., and D. H. M. Groschel. 1985. Leukocyteesterase activity and nitrite test as arapidscreen forsignificant bacteri-uria. Am. J. Clin. Pathol. 83:84-87.

17. Pennie, R. A., R. D. Pearson, and I. T. McAuliffe. Enteric parasitic infections. In R. L. Guerrant, M. A. de Souza, and M. K. Nations(ed.), At theedgeofdevelopment: health crises inatransitionalsociety,in press.OxfordUniversityPress, New York.

18. Pickering, L. K.,H. L. DuPont, J. Olarte, R. Conklin, and C. Ericsson. 1977. Fecal leukocytes in enteric infections. Am. J. Clin. Pathol. 68:562-565.

19. Pryzwansky, K. B., L. E. Martin, and J. K. Spitznagel. 1978. Immunocytochemical localizationof myeloperoxidase, lactofer-rin, lysozyme and neutral proteases in human monocytes and neutrophilic granulocytes. J. Reticuloendothel. Soc. 24:295-309.

20. Speelman, P., R. McGlaughlin, I. Kabir, and T. Butler. 1987. Differential clinical features and stoolfindingsinshigellosis and amoebic dysentery. Trans. R. Soc. Trop. Med. Hyg. 81:549-551.

21. Stoll, B. J., R. I. Glass, M.I.Huq, M. U. Khan, H. Banu, and J. Holt. 1982. Epidemiologic and clinical features of patients infected with shigella who attended a diarrheal disease hospital inBangladesh. J. Infect. Dis. 146:177-183.

22. Tenovuo, J. 1989. Nonimmunoglobulin defense factor in human saliva. In J. Tenovuo (ed.), Human saliva: clinical chemistry andmicrobiology. CRC Press, Inc., Boca Raton, Fla. 23. Williams, E. K., J. A. Lohr, and R. L. Guerrant. 1986. Acute

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References

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